Methods for multiplexing amplification reactions

ABSTRACT

A two-step multiplex amplification reaction includes a first step which truncates the standard initial multiplex amplification round to “boost” the sample copy number by only a 100-1000 fold increase in the target. Following the first step the product is divided into optimized secondary single amplification reactions, each containing one of the primer sets that were used previously in the first or multiplexed booster step. The booster step can occur using an aqueous target nucleic acid or using a solid phase archived nucleic acid. In particular, nucleic acid sequences that uniquely identify  E. Coli  were identified using the multiplex amplification method.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 11/176,795 filed Jul. 7, 2005, and entitled “Methods and Devices forMultiplexing Amplification Reactions,” which is a divisional applicationof U.S. patent application Ser. No. 10/441,158 filed May 19, 2003 andentitled “Methods and Devices for Multiplexing Amplification Reactions”(now U.S. Pat. No. 7,087,414), which is a continuation-in-part of U.S.patent application Ser. No. 09/589,560 filed Jun. 6, 2000, and entitled“Methods and Devices for Multiplexing Amplification Reactions,” (nowU.S. Pat. No. 6,605,451), all of which applications are incorporatedherein in their entireties by this reference.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention pertains to a two-step multiplex amplificationreaction wherein the first step truncates a standard multiplexamplification round to “boost” the sample copy number by only a 100-1000fold increase in the target. Following the first step of the presentinvention, the resulting product is divided into optimized secondarysingle amplification reactions, each containing one of the primer setsthat were used previously in the first or multiplexed booster step. Inparticular, nucleic acid sequences that uniquely identify E. Coli wereidentified using the multiplex amplification method.

2. Description of the State of the Art

Nucleic acid hybridization assays are based on the tendency of twonucleic acid strands to pair at complementary regions. Presently,nucleic acid hybridization assays are primarily used to detect andidentify unique DNA and RNA base sequences or specific genes in acomplete DNA molecule in mixtures of nucleic acid, or in mixtures ofnucleic acid fragments.

Since all biological organisms or specimens contain nucleic acids ofspecific and defined sequences, a universal strategy for nucleic aciddetection has extremely broad applications in a number of diverseresearch and development areas as well as commercial industries. Theidentification of unique DNA or RNA sequences or specific genes withinthe total DNA or RNA extracted from tissue or culture samples mayindicate the presence of physiological or pathological conditions. Inparticular, the identification of unique DNA or RNA sequences orspecific genes, within the total DNA or RNA extracted from human oranimal tissue, may indicate the presence of genetic diseases orconditions such as sickle cell anemia, tissue compatibility, cancer andprecancerous states, or bacterial or viral infections. Theidentification of unique DNA or RNA sequences or specific genes withinthe total DNA or RNA extracted from bacterial cultures or tissuecontaining bacteria may indicate the presence of antibiotic resistance,toxins, viruses, or plasmids, or provide identification between types ofbacteria.

The potential for practical uses of nucleic acid detection was greatlyenhanced by the description of methods to amplify or copy, withfidelity, precise sequences of nucleic acid found at low concentrationto much higher copy numbers, so that they are more readily observed bydetection methods.

The original amplification method is the polymerase chain reactiondescribed by Mullis, et al., in U.S. Pat. Nos. 4,683,195; 4,683,202 and4,965,188, all of which are specifically incorporated herein byreference. Subsequent to the introduction of PCR, a wide array ofstrategies for amplification has been described. See, for example, U.S.Pat. No. 5,130,238 to Malek, entitled “Nucleic Acid Sequence BasedAmplification (NASBA)”; U.S. Pat. No. 5,354,668 to Auerbach, entitled“Isothermal Methodology”; U.S. Pat. No. 5,427,930 to Buirkenmeyer,entitled “Ligase Chain Reaction”; and, U.S. Pat. No. 5,455,166 toWalker, entitled “Strand Displacement Amplification (SDA),” all of whichare specifically incorporated herein by reference.

In general, diagnosis and screening for specific nucleic acids usingnucleic acid amplification techniques has been limited by the necessityof amplifying a single target sequence at a time. In instances where anyof multiple possible nucleic acid sequences may be present, performingmultiple separate assays by this procedure is cumbersome and timeconsuming. For example, the same clinical symptoms generally occur dueto infection from many etiological agents and therefore requiresdifferential diagnosis among numerous possible target organisms. Cancerprognosis and genetic risk is known to be due to multiple genealterations. Genetic polymorphism and mutations result from alterationsat multiple loci and further demand determination of zygosity. In manycircumstances the quantity of the targeted nucleic acid is limited sothat dividing the specimen and using separate repeat analyses is oftennot possible. There is a substantial need for methods enabling thesimultaneous analysis of multiple gene targets for the same specimen. Inamplification-based methodologies, such methods are referred to as“multiplex reactions.”

Chamberlain, et al., (Nucleic Acid Research, (1988) 16:11141-11156)first demonstrated multiplex analysis for the human dystrophin gene.Specific primer sets for additional genetic diseases or infectiousagents have subsequently been identified. See, Caskey, et al., EP364,255A3; Caskey, et al., U.S. Pat. No. 5,582,989; and Wu, et al., U.S.Pat. No. 5,612,473 (1997). The strategy for these multiplex reactionswas accomplished by careful selection and optimization of specificprimers. Developing robust, sensitive and specific multiplex reactionshave demanded a number of specific design considerations and empiricoptimizations. See, Edwards and Gibbs, PCR Methods Applic., (1994)3:S65-S75; Henegariu, et al., Biotechniques, (1997) 23:504-511. Thisresults in long development times and compromises reaction conditionsthat reduce assay sensitivity. Because each multiplex assay requiresrestrictive primer design parameters and empirical determination ofunique reaction conditions, development of new diagnostic tests is verycostly.

A number of specific problems have been identified that limit multiplexreactions. Incorporating primer sets for more than one target requirescareful matching of the reaction efficiencies. If one primer amplifiesits target with even slightly better efficiency, amplification becomesbiased toward the more efficiently amplified target resulting ininefficient amplification of other target genes in the multiplexreaction. This is called “preferential amplification” and results invariable sensitivity and possible total failure of one or more of thetargets in the multiplex reaction. Preferential amplification cansometimes be corrected by carefully matching all primer sequences tosimilar lengths and GC content and optimizing the primer concentrations,for example by increasing the primer concentration of the less efficienttargets. One approach to correct preferential amplification is toincorporate inosine into primers in an attempt to adjust the primeramplification efficiencies (Wu, et al., U.S. Pat. No. 5,738,995 (1998)).Another approach is to design chimeric primers. Each primer contains a3′ region complementary to sequence-specific target recognition and a 5′region made up of a universal sequence. Using the universal sequenceprimer permits the amplification efficiencies of the different targetsto be normalized. See, Shuber, et al., Genome Research, (1995)5:488-493; and U.S. Pat. No. 5,882,856. Chimeric primers have also beenutilized to multiplex isothermal strand displacement amplification(Walker, et al., U.S. Pat. Nos. 5,422,252, 5,624,825, and 5,736,365).

Since multiple primer sets are present, multiplexing is frequentlycomplicated by artifacts resulting from cross-reactivity of the primers.In an attempt to avoid this, primer sequences are aligned using computerBLAST or primer design programs. All possible combinations must beanalyzed so that as the number of targets increases this becomesextremely complex and severely limits primer selection. Even carefullydesigned primer combinations often produce spurious products that resultin either false negative or false positive results. The reactionkinetics and efficiency is altered when more than one reaction isoccurring simultaneously. Each multiplexed reaction for each differentspecimen type must be optimized for MgCl₂ concentration and ratio to thedeoxynucleotide concentration, KCl concentration, Taq polymeraseconcentration, thermal cycling extension and annealing times, andannealing temperatures. There is competition for the reagents inmultiplex reactions so that all of the reactions plateau earlier. As aconsequence, multiplexed reactions in general are less sensitive thanthe corresponding simplex reaction.

Another consideration to simultaneous amplification reactions is thatthere must be a method for the discrimination and detection of each ofthe targets. Generally, this is accomplished by designing the amplifiedproduct size to be different for each target and using gelelectrophoresis to discriminate these. Alternatively, probes or the PCRproducts can be labeled so as to be detectable by spectroscopic,photochemical, biochemical, immunochemical, or chemical means. Forexample, using multiple fluorescent dyes incorporated with aself-quenching probe design amplification can be monitored in real time.See, Livak, et al., U.S. Pat. Nos. 5,538.848 and 5,723,591; and DiCesare, U.S. Pat. No. 5,716,784. The number of multiplexed targets isfurther limited by the number of dye or other label moietiesdistinguishable within the reaction. As the number of differentfluorescent moieties to be detected increases, so does the complexity ofthe optical system and data analysis programs necessary for resultinterpretation. Another approach is to hybridize the amplified multiplexproducts to a solid phase then detect each target. This can utilize aplanar hybridization platform with a defined pattern of capture probes(Granados, et al., U.S. Pat. No. 5,955,268), or capture onto a beadsetthat can be sorted by flow cytometry (Chandler, et al., U.S. Pat. No.5,981,180).

Due to the summation of all of the technical issues discussed, currenttechnology for multiplex gene detection is costly and severely limitedin the number and combinations of genes that can be analyzed. Generally,the reactions multiplex only two or three targets with a maximum ofaround ten targets. Isothermal amplification reactions are more complexthan PCR and even more difficult to multiplex. See, Van Deursen, et al.,Nucleic Acid Research, (1999) 27:e 15.

There is still a need, therefore, for a method which permitsmultiplexing of large numbers of targets without extensive design andoptimization constraints. There is also a further need for a method ofdetecting a significantly larger number of gene targets from a smallquantity of initial target nucleic acid.

Coliform bacteria are introduced into water through either animal orhuman fecal contamination. Monitoring their levels is mandated todetermine the microbiological quality of water. The standards forpotable water include less than one total coliform in 100 milliliterspotable water (Title 40, Code of Federal Regulations (CFR), 1995 rev,Part 141, National Primary Drinking Water Regulations). The coliformgroup of organisms includes bacteria of the Escherichia, Citrobacter,Klebsiella, and Enterobacter genera. However, Escherichia coli is thespecific organism indicative of fecal contamination, since the othermembers of the coliform family can be found naturally in theenvironment. Current water testing methods detect coliforms as a groupso that positive results must be confirmed to be E. coli usingadditional methods. The slow turnaround time for traditional culturedetection and confirmation methods (days) results in delays in detectingcontamination as well as in determining when the water is safe forredistribution or use. Accordingly, there is a need for a rapidmonitoring assay specific for E. coli.

Traditional methods for detecting coliform bacteria rely upon culturingon a medium that selectively permits the growth of gram-negativebacteria and differentially detects lactose-utilizing bacteria (VanPoucke, et al. Appl. Environ. Microbol. (1997) 63(2):771-4; StandardMethods for the Examination of Water and Wastewater, 19^(th) ed.,American Public Health Association, 1995). Since 1880, coliforms havebeen utilized as an indicator organism for monitoring themicrobiological quality of drinking water. However, there are recognizeddeficiencies (Van Poucke, supra). This includes maintaining theviability of bacteria between the time of collection and enumeration,and the existence of chlorine stressed viable but non-culturablebacteria. False negatives can occur due to suppression of coliforms byhigh populations of other organisms or E. coli strains that are unableto ferment lactose (Edberg, et al. Appl Environ Microbiol. (1990)56(2):366-9), and false positives occur due to other organisms thatferment lactose. Culture methods take 24-48 hours for initial coliformenumeration with an additional 24 hours for E. coli confirmation.

Escherichia coli is a member of the family Enterobacteriaceae and assuch, shares much of its genomic sequence with other members of thisfamily (Lampel, et al. Mol. Gen Genet. (1982) 186(1):82-6; Buvinger, etal. J Bacteriol. (1985) September; 163(3):850-7). For many purposes, itwould be useful to specifically identify E. coli in the presence ofother organisms, including members of the same family. However, becauseof the close conservation of sequence between E. coli and otherEnterobacteria, amplification primers specific for E. coli are difficultto design.

Although there are gene-based methods described in the art for thedetection of certain subsets of the coliform group, only a few of theseclaim to detect only E. coli. There are a number of studies that confirmcoliform detection using uidA gene. Lupo et al. (J. Bacteriol. (1970)103:382-386) detected uidA in 97.7% of 435 E. coli isolates, half fromtreated water and half from raw water. Graves and Swaminathan(Diagnostic Molecular Microbiology, (1993) Persing et. al., eds, ASM, p.617-621) detected 100% of 83 confirmed environmental E. coli isolatesusing a uidA probe. Another study (Bej, et al. Appl Environ Microbiol.(1991) 57(4):1013-7) utilized uidA to detect 97% of 116 E. coliisolates. However, specificity studies investigating potentiallycross-reactive organisms confirm that uidA probes detects both E. coliand some Shigella spp. (Bej, et al. (1991) supra; Green et. al., J.Microbiol. Methods (1991) 13:207-214; Rice et. al., J. Environ. Sci.Health A30:1059-1067, 1995).

Total coliforms can be detected using the lacZ gene that codes forbeta-galactosidase (Bej, et al., Appl. Environ. Microbiol. (1990)56(2):307-14; Bej, et al, Appl. Environ. Microbiol. (1991)57(8):2429-32). Utilizing PCR amplification methods, Bej demonstratedlimits of detection of 1-5 CFU in 100 ml of water. Atlas, et. al.disclose lacZ DNA sequences that identify coliform species of the generaEscherichia, Enterobacter, Citrobacter, and Klebsiella (U.S. Pat. No.5,298,392).

Although Min and Baeumner (Anal. Biochem. (2002) 303:186-193) disclosesequences of the heat shock protein gene clpB, their publication onlyshows specificity compared to non-coliform genera and does not includecross-reaction data for other coliforms.

SUMMARY OF THE INVENTION

Accordingly, one aspect of this invention provides a method that permitsthe multiplex amplification of multiple targets without extensive designand optimization constraints. More specifically, this inventioncomprises a two-step multiplex amplification reaction wherein the firststep truncates a standard multiplex amplification round therebyresulting in a product having a boosted target copy number whileminimizing primer artifacts. The second step divides the resultingproduct achieved in the first step into optimized secondary singleamplification reactions, each containing one of the primer sets thatwere used previously in the first step.

This invention further provides a method that enables amplification ofmultiple targets (or multiple amplification assays) from limitedquantity specimens with very low nucleic acid copy number.

This invention further provides a diagnostic kit that allows the user toperform amplification of multiple targets without extensive design andoptimization constraints and to amplify targets from limited quantityspecimens with very low nucleic acid copy number.

This invention further discloses specific nucleic acid sequences thatare unique to E. coli and which are located on the LacZ gene of E. coli.Accordingly, another aspect of this invention provides a method ofdetecting a single CFU of E. coli. More specifically, this inventionprovides a method of utilizing these specific sequences to detect 1 CFUof E. coli following NASBA amplification reactions without the need forlong culture enrichment. This invention further includes a method ofusing IPTG induction to increase sensitivity in detecting the unique E.coli sequences disclosed herein.

Additional advantages and novel features of this invention shall be setforth in part in the description that follows, and in part will becomeapparent to those skilled in the art upon examination of the followingspecification or may be learned by the practice of the invention. Theadvantages of the invention may be realized and attained by means of theinstrumentalities, combinations, compositions, and methods particularlypointed out in the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and form a part ofthe specification, illustrate the preferred embodiments of the presentinvention, and together with the description serves to explain theprinciples of the invention.

In the Drawings:

FIG. 1 is a diagrammatic illustration of the limited multiplexamplification method of the present invention.

FIG. 2A is an illustration of an agarose gel showing the results of theexperiment in Examples 1 and 2.

FIG. 2B is an illustration of an agarose gel showing the results of theexperiment in Examples 1 and 2.

FIG. 2C is an illustration of an agarose gel showing the results of theexperiment in Examples 1 and 2.

FIG. 2D is an illustration of an agarose gel showing the results of theexperiment in Examples 1 and 2.

FIG. 3 is an illustration of an agarose gel showing the results of theexperiment in Example 1-3.

FIG. 4 shows the detection results of a lateral flow assay of uninducedand induced E. coli culture.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

As discussed above, it would be tremendously useful if a method anddiagnostic kit could be devised to multiplex multiple nucleic acidtargets without the necessity of complex design constraints andextensive optimizations. The methods and diagnostic kit of the presentinvention generally involve the use of common multiplex amplificationmethods and reagents and are more specifically derived from thesurprising discovery that if the nucleic acid sample to be analyzed isfirst pre-amplified so as to merely “boost” the samples copy numberslightly, then the resulting product may be split into as manysubsequent analysis reactions as required, and thereby circumventing thecommon limitations of standard multiplexing technology.

The present invention describes a novel method for boosting the amountof nucleic acid obtained from a broad range of biological samples fordistribution among large numbers of individual analyses. It preferablyinvolves the utilization of archived nucleic acid, bound irreversibly toa solid-phase material as the starting material. See U.S. applicationSer. No. 09/061,757 and corresponding international application WO98/46797, each of which is specifically incorporated herein byreference. The copy number of the irreversibly bond nucleic acid is then“boosted” by running a limited multiplex amplification reaction. Thelimited multiplex amplification reaction is a truncated version of anywell known amplification methods such as, but not limited to PCR,RT-PCR, NASBA, SDA, TMA, CRCA, Ligase Chain Reaction, etc. Usingarchived nucleic acid has the advantage that multiple sequential roundsof the present invention may be performed. Alternatively, nucleic acidsthat are not bound but in aqueous solution may also be used. In thisinstance nucleic acid is obtained from the desired biological sample bya number of common procedures. These include phenol-chloroform and/orethanol precipitation (Maniatis, et al., Molecular Cloning; A LaboratoryManual), high salt precipitation (Dykes, Electrophoresis (1989)9:359-368), chex and other boiling methods (Walsh, et al.,Biotechniques, (1991) 10:506-513 and other solid phase binding andelution (Vogelstein and Gillespie, Proc. Nat. Acad. Sci. USA, (1979)76:615-619, etc. Output from these initial rounds of limitedamplifications is distributed into the several single analyses.

As discussed above, it is desirable to be able to detect a single CFU ofE. coli. Current methods for obtaining this level of sensitivitygenerally require culturing the organism overnight in order to reachdetectable cell numbers. Both the lacZ beta-galactosidase (Bej, et al.Appl Environ. Microbiol. (1991) 57(8):2429-32; Sheridan, et al. Appl.Environ. Microbiol. (1998) 64:1313-1318) and the uidA beta glucuronidaseenzymes (Vaitilingom, et al., Appl. Environ. Microbiol. (1998)64:1157-1160; Berg and Fiksdal, Appl. Environ. Microbiol. (1988)54:2118-2122; Tryland I, et al. Appl. Environ. Microbiol. (1998)64(3):1018-23) have been shown to be readily inducible usingenvironmental isolates. Lactose induction of membrane filter collectedenvironmental isolates was shown to increase enzyme activity as much as1000-fold resulting in detection limit of 100 CFU per 100 ml within 15minutes of collection (Davies, et al., Lett. Appl. Mlcrobiol. (1995)21(2):99-102).

This invention provides a unique strategy to increase sensitivity byinducing the transcription of multiple mRNA copies within the E. colicell in order to more rapidly reach detectable levels following NASBAamplification. Detection of a single CFU of E. coli is accomplished bydiluting the bacteria suspended in water into an induction media withisopropyl β-D-thiogalactopyranoside (IPTG) and incubating for about 2-6hours to allow for mRNA transcription as described in detail in Example3. The cells are lysed and released RNA bound to Xtra Amp™ tubes usingthe package insert directions (see also U.S. Pat. No. 6,291,166, whichis specifically incorporated herein by reference). The solid phasecaptured RNA is amplified directly using the E. coli target recognitionsequences disclosed herein that have been modified for NASBAamplification and lateral flow detection as described in U.S. Pat. No.5,989,813 and U.S. patent application Ser. No. 09/705,043, each of whichare specifically incorporated herein by reference.

In one preferred embodiment of the present invention, a samplecontaining tissue, cells or other biological material is treated in thepresence of the solid phase binding material to release the nucleic acidcontained in that sample. The solid phase archiving material allowsamplification without elution of the bond nucleic acid. Once the samplenucleic acid is bound to the solid phase, the solid phase material iswashed to remove lysis buffer conditions, and to prepare foramplification conditions. A reaction mixture appropriate to theamplification method is added to the solid phase or allowed to contactthe solid phase. The number of primer pairs used according to thepresent invention may be any number greater than two, however, since thestandard multiplexing reaction conditions and designs become moredifficult and less effective as the number of primers used increases thepresent invention is most helpful as the number of primers utilized isover five.

This reaction mixture contains the amplification primers for severalindependent amplifications. This is similar to standard multiplex PCRwith the following exceptions: first, in the preferred embodiment, theprimer concentrations will be very low. The effective primerconcentrations should be limited to allow only a few logs ofamplification but definitely the primer concentration should beexhausted before reaction plateau is reached. Second, the number ofamplification cycles should also be minimized. The goal of this firstphase of multiplex amplification or “Booster Amp” is to allow only asufficient amount of amplification to proceed in order to allow theresultant reaction mix to be split up and redistributed into at leastthe same number of subsequent simples amplification reactions as thereare independent primer pairs in the first amplification.

This initial round of amplification, or Booster Amp should only proceedinto early logarithmic phase of the amplification, and in no instanceproceed until reaction plateau is reached. Once the Booster Amp iscomplete, the resultant reaction mix with the multiple amplificationspecies is removed from contact with the solid phase and distributedinto several secondary amplifications. These amplifications could beequal to or greater than the number of primer pairs employed in thefirst Booster Amp. Each of these secondary amplification reactions willcontain only one primer pair. This primer pair may be identical to oneof the primer pairs in the Booster Amp or may be “nested” within one ofthe primer pairs of the Booster Amp. Either way, each secondaryamplification reaction will use the input material from the Boosteramplification as a target source. In the secondary amplification, normalamounts of primer will be used, and amplification will be allowed toproceed normally. A detection system for normal detection of a singleamplification product such as, but not limited to radioactive isotopes,or visual markers such as biotin may be included in the secondaryamplification.

In the preferred embodiment the reaction takes place in the presence ofa solid phase material as discussed previously. The advantage of this isthat the original solid phase material with the bond nucleic acid may berinsed following the first Booster Amp and re-initialized for a second,subsequent Booster Amp employing a new mix of amplification primers. Theprocess is then repeated through an additional secondary amplification.The entire process can be repeated for numerous rounds. Consequently, inthe event the quantity of nucleic acid sample being analyzed is low, theanalysis may be performed as often and as thoroughly as need be.Alternatively, the Booster Amp step may be performed in aqueouscondition where the nucleic acid is unbound.

FIG. 1 illustrates the concept for microwell based PCR (but can apply toany primer-based amplification method utilizing a polymerase, such asbut not limited to DNA polymerase, RNA polymerase, transcriptase, or Qβreplicase in any form of an appropriate container). A chip or cardcontaining the reaction wells or chambers (not shown) is contained in adevice capable of performing the correct thermocycling. Lysed sample isintroduced into extraction chamber 10, where the freed nucleic acid canbind to the solid phase material within extraction chamber 10. Thechamber 10 having bound the sample is incubated is step 12 for a shortperiod (10-20 minutes). An aqueous buffer, preferably PCR buffer is thenused in washing step 14. The wash 14 removes lysate and initializes thechamber 10′ in step 16 for PCR conditions. The first multiplex PCRreaction mixture (containing multiplexed primers, PCR buffer, and TaqPolymerase) is introduced to the chamber 10 and cycled in step 18. Themultiplex products 20′ should be sub-detectable, but amplified to alevel no greater than the plateau of the reaction and preferably in therange of 100 to 1000-fold. The Booster PCR reaction 20′ is then splitinto the secondary PCR chambers 22′. Reaction mixtures (having a singleprimer pair) for each of the simplex, secondary PCR reactions (notshown) are introduced previously, or at this point. Cycling is performedon the chip or card to allow the secondary PCR reactions 22′ to proceedto completion. The initial sample chamber is now empty, and it can bewashed at step 24 and re-initialized for a second round ofBooster/Secondary PCRs.

This invention includes nucleic acid sequences that are substantiallyhomologous to the SEQ ID NO. 99. By substantially homologous it is meanta degree of primary sequence homology in excess of 70%, most preferablyin excess of 80%.

This invention reveals a robust and simple method for multiplexamplification of large numbers of gene targets. The invention teachesthat preferably 2-40 or more primer sets can be combined to permitmultiplex amplification if they are in low concentration and limited toan initial amplification round that results in only a 100-1000 foldincrease in target. However, it should be understood that any number ofprimer sets greater than two may be used according to the presentinvention. This has been designated as a “booster round” or booster ampand can occur using an aqueous target nucleic acid or using solid phasearchived nucleic acid. As discussed above, the advantage of usingarchived material is that multiple booster rounds can be performed fromthe same archived specimen. For example, performing five, 20-targetbooster rounds from archived nucleic acid would permit the analysis of100 different genes. Following each booster round the amplificationproduct is diluted into optimized secondary single PCR reactions, eachcontaining one of the primer sets that were multiplexed in the boosterreaction. These simplex reactions can be optimized for maximumsensitivity and each requires only one method of detection, for examplesingle dye homogeneous detection. The invention enables multiplexingwithout extensive optimization and is robust enough to permit randomselection of the primers to be multiplexed.

The invention overcomes the usual technical problems of multiplexing. Bylimiting the multiplexed cycles, preferential amplification andcross-reaction of the multiple primers is minimized. Only the single PCRreactions need to be optimized. The simplex reaction has maximumsensitivity since reagent competition does not occur. By using thesimplex PCR the detection probe does not have to be multiplexed. Thepotential to randomly combine the multiplexed primers provides formaximum flexibility and cost effectiveness since this allows customselection of the targets to be multiplexed. Frequently, the targets thatneed to be multiplexed can vary for a particular geographic location,laboratory, type of patient, or type of specimen. Since archived nucleicacid can be reanalyzed the multiplex can be designed in a logicalalgorithm. For example, in booster reaction detection set number one,identify the most frequently known mutations. Then only if these are notdetected is it necessary to perform additional multiplexes for the morerare mutations. This enables economical yet comprehensive geneticanalysis.

The invention is further illustrated by the following non-limitedexamples. All scientific and technical terms have the meanings asunderstood by one of ordinary skill in the art. The specific exampleswhich follow illustrate the various multiplexing amplification methodsthat the present invention may be adapted to work with and are not to beconstrued as limiting the invention in sphere or scope. The methods maybe adapted to variation in order to function with other commonly usedmultiplex amplification methods embraced by this invention but notspecifically disclosed. Further, variations to the methods to producethe same results in somewhat different fashion will be evident to oneskilled in the art.

All temperatures are understood to be in degrees Centigrade (° C.) whennot specified. Melting temperatures (Tm) of the primers were estimatedusing the generally accepted mathematical calculation based upon theformula Tm=81.5+16.6×log (Na⁺) (41×(#G+#C)/length)−500/length.Amplification techniques, including multiplexing amplificationtechniques, are now sufficiently well known and widespread so as to beconsidered routine. All polymerase enzymes and nucleotides can bepurchased from PE (Biosystems, Foster City, Calif.). PCR was carried outin a buffer containing (50 mM KCl, 10 mM Tris, pH 8.3, 2.0 mM Mg²⁺) for30 cycles of 1 minute at 94° C., for 2 minutes at 55° C. and at 72° C.with a 5 second increment added to the 72° C. elongation step at everycycle. This procedure was carried out in a DNA Thermal Cycler(Perkin-Elmer Cetus catalog No. N8010150).

Example 1 Multiplex Amplification Incorporating Booster PCR andArchiving 1. Primer Design:

The Xtra Amp™ Extraction Kit (Xtrana, Inc.) provides an innovativesystem for nucleic acid extraction in which the nucleic acid remainsbound in the extraction tube and can be directly amplified by PCR inthis same tube. The unique principle underlying this system lies in theproprietary nucleic acid binding matrix, Xtra Bind™. Xtra Bind™ is anon-silica matrix which stably and irreversibly binds both DNA and RNA.The Xtra Amp™ kit contains 96 (as 1×8 strips) 0.2 mL sizemicrocentrifuge tubes coated with the Xtra Bind™ matrix, cell lysisbuffer and wash buffer. The kit is commercially available currently forextraction of genomic DNA from, whole blood (manufactured anddistributed for Xtrana, Inc. by ANSYS Diagnostics, Lake Forest, Calif.).For demonstrating Xtra Plex feasibility, the Xtra Amp™ Blood kit waschosen as the extraction platform. For the PCR multiplexing experimentsit was decided to obtain twenty five primer pairs to allow their use invarious groupings. Three primer sets from the Lifecodes Corporation(Stanford, Conn.) HLA Primers (HLA-A: Primer A1 (catalog No. 164011);Primer A2 (catalog No. 164012); HLA-B: Primer B1 (catalog No. 165011),Primer B2 (catalog No. 165012; DRβ: Primer A (catalog No. 160031);Primer B (catalog No. 160032) were added to twenty three in-house primersets, shown below in Table 1, that were designed for human gene targetsto make the total of twenty five sets. The genes targeted are asfollows: Human cytoplasmic beta-actin gene (accession M10277); Homosapiens interleukin 2 precursor (IL2) gene (accession J00264); Humangrowth hormone gene (HGH-N) (accession M13438); Humanglyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene (accessionJ04038); Homo sapiens dystrophin (DMD) gene (accession AF214530); Homosapiens G protein-coupled receptor 57 (GPR57) gene (accession AF112461);Human galactokinase (GALK1) gene (accession L76927); Homo sapiens MHCclass I region (accession AF055066). The primer pairs were designedusing the primer design software OLIGO 5.0 (Molecular Biology Insights).The sizes of the amplification products are designed to allowdiscrimination by agarose gel electrophoresis. No special considerationswere given to normalizing the melting temperatures of the primer pairs,or to eliminating structural interactions between primers from differentpairs. PCR primer design considerations such as avoiding hairpins anddimers between the forward and reverse primers of each set wereaccommodated in the design as for any typical PCR primer design. Theresultant primers had melting temperatures ranging from 49° C. to 72° C.When examining the dimer formation potential between primers fromdifferent pairs, some serious dimer potential was seen in some cases.This was largely ignored since this approach was believed to be able toovercome such potential artifacts and it was considered important todemonstrate this. The primers and sequences are shown in Table 1.

TABLE 1 Primer SEQ ID Amplicon Pair NO: Sequence Name TM Length Group 1 1 1 CGAGGCCCAGAGCAA HBAPCR1-FP 58 100 2 GGTGTGGTGCCAGATTT HBAPCR1-RP 57100  2 3 GTTTAATCAGAGCCACA IL2=PCR2-FP 52 187 4 GGAAAGGTAGGTCAAGAIL2 PCR2-RP 54 187  3 5 GTCTTGCATTGCACTAA IL2-OLD-fp 52 257 6TAAATGTGAGCATCCTG IL2-OLD-rp 52 257  4 7 CTGTGGAGGGCAGCTGTGGChGH PCR1-FP 68 450 TT 8 GCGGGCGGATTACCTGAGGT hGH PCR1-RP 68 450 CA  5Lifecodes HLA-A, A1 HLA-A-A1 N/A 910 Lifecodes HLA-A, A2 HLA-A-A2 N/A910 Group 2  6 9 TCAGCAGAGAAGCCTAA IL2-PCR1-FP 54 120 10ATCCCTACCCCATCAT IL2-PCR1-RP 54 120  7 11 CAAAAGTCCTCCCAAAG IL2-PCR3-FP54 197 12 TGCCATCTATCACAATCC IL2-PCR3-RP 55 197  8 13 AAGGGTCATCATCTCTGCGAPDH 15 fp 57 259 14 CTTCCACGATACCAAAGTT GAPDH 15 rp 55 259  9 15CGCTTTAAGTTATTTGTGTC HDYST3-FP 54 400 16 GTTTCCTTTTAAGGGTATTC HDYST3-RP54 400 10 Lifecodes HLA-B, B1 HLA-B-B1 N/A 1100 Lifecodes HLA-B, B2HLA-B-B1 N/A 1100 Group 3 11 17 CATCTACGAGGGGTATG HBAPCR2-FP 57 120 18GCCGTGGTGGTGA HBAPCR2-RP 54 120 12 19 GTTTGCCTTTTATGGTAATAA HBAPCR4-FP55 161 C 20 GTGAGCTGCGAGAA HBAPCR4-RP 54 161 13 21 GAGTCCACTGGCGTCTTCACGAPDH FP 64 233 22 AGGCTGTTGTCATACTTCTC GAPDH RP 58 233 14 23CCACCCCCTTAAAGAAA IL2-PCR-FP 54 346 24 GGCAGGAGTTGAGGTTA IL2-PCR4-RP 57346 15 25 GCGGGGAGGAGGAAAGGAA hGHPCR2-FP 66 500 26 CAGGACACATTGTGCCAAAGhGHPCR2-RP 64 500 GG Group 4 16 27 CCACTATTCGGAAACTT HGP57R1-FP 52 13028 TGTATGGCATAATGACA HGP57R1-RP 49 130 17 29 GAGTCGAGGGATGGCTAGGHDYST1-FP 64 150 T 30 TTCAAAGTGGGATGAGGAG HDYST1-RP 60 150 G 18 31GGACTGCCACCTTCTACC HGKPCR2-FP 62 215 32 GACACCCAAGCATACACC HGKPCR2-RP 59215 19 33 GCAGATGAGCATACGCTGA hGHPCR3-FP 64 600 GTG 34CGAGGGGAAATGAAGAATA hGHPCR3-RP 62 600 CGG 20 Lifecodes DR-β, A DR-β, AN/A 287 Lifecodes DR-β, B DR-β, B N/A 287 Group 5 21 35AGGGGAGGTGATAGCAT HBAPCR3-FP 57 140 36 AAGTTGGGGGACAAAA HBAPCR3-RP 51140 22 37 CCGGTGCCATCTTCCATA HGKPCR1-FP 68 170 38 CCTGCCTTGCCCATTCTTHGKPCR1-RP 68 170 23 39 GAGGGGAGAGGGGGTAA HBAPCR5-FP 62 228 40CGGCGGGTGTGGA HBAPCR5-RP 57 228 24 41 GGCTGCTTTTAACTCTGG GAPDH FP 57 27042 CACTCCTGGAAGATGGTGAT GAPDH RP 64 270 25 43 CTCATTCTCTAGCCAAATCTHDYST2-FP 56 300 44 CCTCGACTCACTCTCCTC HDYST2-RP 62 300 New Primers 2645 CTATCGCCATCTAAGCCCAG HGH PCR4-fp 62 450 46 CTGCCTGCATTTTCACTTCAHGH PCR4-Rp 58 450

2. Sequential Booster Reaction of Solid-Phase Captured Nucleic Acid.

In these multiplexing experiments, (the results of which aredemonstrated in FIGS. 2a-2d ) designed to show feasibility forperforming multiple rounds of multiplexing amplification, each boosteramplification consisted of a PCR reaction with five primer sets. In thefirst 5-plex experiment, Group 2 primers were used. In the secondexperiment group 4 primers were used, and in the third, group 1 primerswere used. In the 20-plex experiment, the primers used were selectedfrom all 5 groups each at one-fiftieth normal concentration (4 nM asopposed to 200 nM). The nucleotide triphosphate, buffer and saltconcentrations were normal. Cycling conditions were chosen as acompromise between the theoretically optimal conditions for each of theprimer pairs (each cycle: 72° C., 30 seconds; 55° C., 1 minute; and 65°C., 3 minutes). The total number of cycles was limited to ten. Theprimers were first mixed in 1:1 ratios to each and then diluted toone-fiftieth of the normal 10× concentration. The PCR reaction mix forthe booster reaction was made up as per normal. The Xtra Amp™ kit(discussed previously) was used with fresh human blood as per the kitprotocol. The booster reactions were placed into the Xtra Amp™ tubes aswould a normal PCR. Following the booster PCR, the reaction mixture wasremoved and diluted 5-fold.

To each simplex, secondary reaction 5 microliters of this dilutedbooster product was added. Each secondary reaction had only one of the 5primer pairs and was set up normally with normal amounts of primer.These PCRs were run for the full 40 cycles. Two cycling profiles (72=C,30 seconds; 65° C., or 1 minute; 72° C., 3 minutes) were used for thesesecondary reactions. The same cycling profile used in the boosterreactions was used for all secondary PCRs. In cases where the primerswere believed to require higher temperatures, additional secondaryreactions were run using higher temperature profiles (each cycle: 72°C., 30 seconds; 65° C., 1 minute; 72° C., 3 minutes).

Next, the Xtra Amp™ tube in which the booster reaction was performed wasrinsed three times with Wash Buffer supplied in the kit. A secondbooster PCR reaction mixture with a different set of five primer pairswas added to the tube. Identical conditions were used as in the firstbooster amplification. The product was removed and diluted as before,and aliquoted into the next group of five secondary, simplex PCRs usingthe same primer pairs as in the second booster PCR. All of thesesecondary reactions were run using the same cycling profile as thebooster, with some primers being additionally run at the higher tempprofile as well. Following this, a third round of booster/secondarysimplex reactions were run with a third grouping of five primers inidentical conditions to the first two rounds.

For comparison, a normal, 40-cycle multiplex PCR was run using each ofthe five primer pair groupings. Each of these had five primer pairs eachin the same groupings as the booster PCRs. These multiplex reactionswere run with half-normal primer concentration for 40 cycles. Theproducts from all fifteen secondary reactions, the high-temp additionalsecondary reactions and the three normal multiplexing PCRs were analyzedby agarose gel electrophoresis.

FIG. 2A demonstrates the results of the first round of the secondary PCRreactions. Five PCRs were used in first round (Group 2: IL2-1; IL2-3;GAPDH 15; HDYST3; HLA-B) booster PCR. The Booster PCR was performed inan Xtra Amp™ tube following extraction of nucleic acids from humanblood. Following booster amplification, product was diluted 1:5, then1:10 volume of this was added to six subsequent reactions in separatenormal PCR tubes. The six subsequent simplex secondary reactionsincluded all five of the Booster PCR systems, with an additional system(HLA-A) whose primers were similar to one of the five (HLA-B). Theadditional PCR served as a secondary PCR control and was expected to benegative. The results shown in FIG. 2A demonstrate that all five PCRsfrom Booster worked in the secondary simplex reactions. In contrast, anormal multiplex PCR (MMX) in which all five primer pairs were run in asimilarly prepared Xtra Amp™ tube for a total of 40 cycles did not workfor all PCR products. (In this and all gel images pictured, thereference ladder is in 100 base-pair increments starting at 100 bp L isladder, nt is no template and t is template).

FIG. 2B demonstrates the results of the second round of the secondaryPCR reaction. The second round of 5-primer pair booster PCR was run inthe same Xtra Amp™ tube as was the first booster. The tube was washedthree times with Xtra Amp™ kit wash buffer prior to the addition of thesecond booster multiplex PCR mix. The next five primer pairs were fromGroup 4: HGP57; HDYST-1; HGK-2; HGH-3, and DRB. Following an identicallyrun booster PCR, the product was diluted 1:5 as before and aliquotedinto the five secondary simplex PCR reactions. These reactions were runat the low temperature cycling range. The results shown in FIG. 2Bdemonstrates that the four reactions worked at this temperature withsome spot contamination in one of the HDYST-1 no-template controls. TheHGP57 and HGH-3 secondary reactions work best at the high temperaturesecondary cycling protocol and were done at that temperature as well. Anormal PCR multiplex (MMX) with these same primers in an Xtra Amp™ tubefailed again.

FIG. 2C demonstrates the results of the third round of the secondary PCRreactions. In this experiment, a third 5-primer pair booster multiplexwas performed in the same Xtra Amp™ tube as was the first two boosterreactions, discussed above, using primer sets from group 1 (HBA PCR 1;IL2 PCR 2; IL2 PCR-old; hGH PCR 1 and HLA-A). Again, three standardwashes were done with the tube between the previous booster PCR and thisone. The standard 40-cycle multiplex with these primers failed withthese primers as well. The results shown in FIG. 2C demonstrates thatsome contamination in the no template controls was seen in the IL2-OLDprimer reaction, and the hGH-1 PCR did not work well with the lowtemperature cycling condition set for the secondary reactions. Both thehGH-1 and HLA-A secondary reactions were run at the high temperaturesecondary cycling condition set (65° C. and 72° C. vs. 55° C. and 65°C.).

The results shown in FIG. 2D demonstrates that in this set of secondaryreactions, the primer sets which appeared to be compromised in the firstsets of secondary reactions were rerun using higher annealing andextension temperatures (65° C. and 72° C. vs. 55° C. and 65° C.,respectively). The contamination issue with IL2-OLD was still apparent(this primer set had been used previously in our lab). The remaining PCRreactions were much more robust.

The results indicate that all of the secondary reactions worked. In onecase where a primer pair had been used extensively in this lab prior tothese experiments (IL2-OLD) there appeared some contamination in the notemplate controls. One primer pair gave weak positive results (HGP-57).Four primer pairs worked better at the higher temperature secondary PCRprofile. One primer pair worked better at higher concentration in thesecondary reaction. These results indicate that the secondary PCRreactions function as normal PCR reactions in which some optimizationwill give better results. The booster PCR was able to functionadequately at less than optimal conditions for the various primer sets.In contrast, the three normal multiplex PCRs that were run as multiplexamplifications for the full 40 cycles failed to show amplificationproducts for all but a few primer sets.

3. Multiplex Capability Enabled by Booster PCR:

Since the two-step PCR approach in which a limited-booster PCR is usedto boost the target copy prior to aliquoting the boosted product intothe individual secondary PCRs worked so well with groups of 5 primerpairs, increasing the number of primer pairs in the booster reaction wasperformed. A 20-primer pair booster reaction was set up using identicalconditions as before, except that in addition to using the Xtra Amp™ kitwith human blood as the sample, a booster PCR was set up in a normal PCRtube using previously extracted human placental DNA as the template. Theprimer set 23 replaced primer set 4 in this experiment. The individualprimers were at 4 nM or one-fiftieth of normal concentration. Thebooster PCR was run for 10 cycles using the lower temperature profile(each cycle: 72° C., 30 seconds; 55° C., 1 minute; 65° C., 3 minutes).The product for this booster reaction was diluted 5-fold and used astemplate for the 20 secondary, simplex PCR reactions. The products fromthese reactions were analyzed by agarose gel electrophoresis as shown inFIG. 3. Although the contrast of the pictures obscures the results insome cases, all reactions gave product at the expected sizes for theaqueous PCRs. In some instances the aqueous reactions gave slightlybetter results than was seen for the reactions whose booster PCR wasdone in Xtra Amp™ tubes. Further optimization of the secondary reactionsshould eliminate those differences. The last four reactions (HGH-3,HLA-A, HLA-B and DRβ were done using the high temperature cyclingcondition set in the secondary reactions).

The results showed that all of the secondary PCRs from the aqueous(non-Xtra Amp) booster worked. Some of the secondary PCRs still needsome minor optimization in order to yield cleaner products, butcorrectly sized product bands were seen for all reactions. Threesecondary reactions from the Xtra Amp™ booster showed very weak product(poorly reproduced on gel photo). Additional optimization of thesecondary reactions should improve the performance. It is noteworthythat even with 40 different primers being present in the booster PCRs,the secondary reactions worked yielding products of the correct size.This indicates that primer-primer artifacts across primer systems thatmay form in the booster reaction, and would most certainly form in atraditional one-step multiplex, do not interfere significantly with thesecondary simplex reactions.

By performing the two-step multiplex of multiplex booster followed bysimplex secondary reactions, large numbers of assays can be done with asingle sample without encountering the confounds associated with normalsingle-step multiplex amplification. Of further note is the fact that,even when using PCR primers that have not been optimized in any way andboosting with a profile that is demonstrably less than optimal for atleast some of the primer sets used, the approach is still robust.

Example 2 Sequential Booster Xtra Plex NASBA Reaction of Solid-PhaseCaptured Nucleic Acid 1. Booster Xtra Plex NASBA First Pass

Several NASBA systems are currently in operation in our lab. From these,ten primer sets as shown in Table 2 were chosen. These were: (1) forEscherichia coli: SZ gene (SZ), SLT1 and 2 genes (SLT1, SLT2), 16Sribosomal RNA (16S), LacZ (LacZ 1 and LacZ 2), and UIDA gene (UIDA); (2)for Listeria monocytogenes: HlyA gene; (3) for Nisseria gonorrhea: 16sribosomal RNA (NG2); and (4) for Chlamydia trachomatis: 16s ribosomalRNA (CT2). Surprisingly, the primers LACZ 1 and LACZ 2 have been shownto be specific to E. coli and can discriminate E. coli from othercoliform species. The 16s ribosomal (16s) primer set is capable ofbinding to and allowing amplification of all gram-negative bacteriaexamined and may be a universal primer set for all gram-negativebacteria.

TABLE 2 SEQ ID ORGANISM NO: GENE^(a) SEQUENCE C. trachomatis 47 CT2: FPAATTCTAATACGACTCACTATAGGGAGAGG TAACCGAAAGGTCCTAAGAT 48 CT2: RPATTGTTTAGTGGCGGAAGGG 49 CT2: 5DP FITC-ACTTGGGAATAACGGTTGGAAA-PO₄ 50ct2: 3DP GCTAATACCGAATGTGGCGATA-Biotin N. gonorrhea 51 NG2: FPAATTCTAATACGACTCACTATAGGGAGAAC TGACATCGTTTAGGGCGTGG 52 NG2: RPAATGCGTAGAGATGTGGAGG 53 NG2: 5DP FITC-AGCCTCCTGGGATAACACTGACG-PO₄ 54NG2: 3DP AGCGTGGGTAGCAAACAGGATTAGA-Biotin E. coli 55 LacZ 1: FPAATTCTAATACGACTCACTATAGGGAGAGG AAACTGCTGCTGGTGTTTTGCTT 56 LacZ 1: RPTTACGGCGGTGATTTTGGCGAT 57 LacZ 1: 5DP FITC-ATCGCCAGTTCTGTATGA-PO₄ 58LacZ 1: 3DP CCGACCGCACGCCGCATCCAGC-Biotin E. coli 59 LacZ 2: FPATTCTAATACGACTCACTATAGGGAGAGGA GAACTGGAAAAACTGCTGCTGG 60 LacZ 2: RPCGTTTACAGGGCGGCTTCGTCT 61 LacZ 2: 5 DP FITC-ATCGCCAGTTCTGTATGA-PO₄ 62LacZ 2: 3DP CCGACCGCACGCCGCATCCAGC-Biotin Coliform Bacteria 63 UIDA: FPAATTCTAATACGACTCACTATAGGGAGA GGAATAGTCTGCCAGTTCAGTTCGTTGT 64 UIDA: RPCAAAGTGTGGGTCAATAATCAGGAA 65 UIDA: 5DP FITC-CTATACGCCATTTGAAGCCGAT-PO₄66 UIDA: 3DP GTCACGCCGTATGTTATTGCCG-Biotin E. coli 0157: H7 67 SZ: FPTTGTTAGCGTTACGTTTCCCTCT 68 SZ: RP AATTCTAATACGACTCACTATAGGGGAGAGGATAATACCAAATCAGGTTTTCCATTGA 69 SZ: 5DPFITC-CGATGATGCTACCCCTGAAAAACT-PO₄ 70 SZ: 3DPGAGAATGAAATAGAAGTCGTTGTT-Biotin E. coli 0157: H7 71 SLT 1: FPGTTTGCAGTTGATGTCAGAGG 72 SLT 1: RP ATTCTAATACGACTCACTATAGGGAGAGGAACGTGGTATAGCTACTGTC 73 SLT 1: 5DP FITC-ATCTACGGCTTATTGTTGAACGAAA-PO₄ 74SLT 1: 3DP TTTTATCGCTTTGCTGATTTTTCAC - Biotin E. coli 75 SLT 2: FPTTGCTGTGGATATACGAGGG 76 SLT 2: RP ATTCTAATACGACTCACTATAGGGAGAGGAGAGTGGTATAACTGCTGTC 77 SLT 2: 5DP FITC-TTTTGACCATCTTCGTCTGATTATT-PO₄ 78SLT 2: 3DP GTTAATACGGCAACAAATACTTTCT-Biotin Universal 79 16S: FPAATTCTAATACGACTCACTATAGGGAGAGGACC TTGTTACGACTTCACCCCAG 80 16S: RPTACACACCGCCCGTCACACCAT L. monocytogenes 81 HlyA: FPAATTCTAATACGACTCACTATAGGGAGAACCTTT TCTTGGCGGCACA 82 HlyA: RPGTCCTAAGACGCCAATCGAA 83 HlyA: 5DP FITC-AACACGCTGATGAAATCTATAAGTATA-PO₄84 HlyA: 3DP GTATTAGTATACCACGGAGATGCAGTG-Biotin ^(a)primer and probesequences for NASBA (FP = forward primer, RP - reverse primer 5DP =5′-lateral flow detection probe, and 3DP = 3′- lateral flow detectionprobe)

Various cell extracts and artificial RNA templates were pooled(extracted RNAs, RNA runoff material, and RNA product from previousNASBA reactions). Twenty microliters of each were combined then diluted1:1 with water. Thirty microliters of this mix was added to Xtra Amp™tubes. To this was then added an equal volume of 2×LiCl lysis buffer.The solution was mixed and let sit for 20 minutes at room temperature.After 3 washes with LiCl wash buffer, the tubes were ready for NASBA.

Two sequential booster groups of 5 primer pairs each were then run intandem. The booster NASBA reactions were run with normal reactioncomponents and concentrations except that the primers were pooled andrun at one-fiftieth of normal concentration (4 nM instead of 200 nM).Two booster reactions were set up in parallel and run for differenttimes. The first was run for 15 minutes at 40° C., and the second for 30minutes at 40° C. The reactions were terminated and the products removedfrom the tubes. The tubes were washed for the next round. The productswere diluted 10-fold and used as template for the subsequent secondarysimplex reactions. These reactions were run normally and analyzed byagarose gel electrophoresis and lateral flow where possible. The secondgroup of five primer sets was then run identically in the same Xtra Amp™tubes as were used in the first group booster. The procedure frombooster to secondary simplex amplifications was identical. The groupingsof the primer pairs were as follows: Group 1: SZ, SLT2, HlyA, CT2, andLacZ2; Group 2: SLT1, 16S, NG2, LacZ1, and UIDA1.

The results indicated that four of the five secondary reactions from thefirst booster reaction worked as indicated either by agarose gelelectrophoresis or lateral flow detection. The system that failed. E.coli SLT-2, is a system that has a history of inconsistent performancein our laboratory. All five of the secondary reactions from the secondbooster reaction worked. The gel results and lateral flow (Gerdes, U.S.Pat. No. 5,989,813 incorporated herein by references) results variedslightly. This variance has been seen with these systems previously andis indicative of sub-optimal conditions. Thus, although the reactionconditions require further optimization for full performance in theBooster Xtra Plex paradigm, the results are extremely encouraging andnoteworthy in that they were so positive with completely untested andhighly preliminary conditions.

2. Multiplex Capability Enabled by Booster NASBA:

Multiplexing isothermal amplification reactions is even more difficultthan multiplexing PCR reactions. However there are applications in whichan isothermal amplification of multiple targets would be the method ofchoice if that were possible. By employing the booster strategydeveloped for PCR but with NASBA primers this has been accomplished. Ashort booster NASBA reaction in which the ten primer pairs targetinggenes products from diverse organisms was performed as above, but withall ten present in the same reaction. The booster NASBA was run with theHlyA NASBA condition set using 1:50 diluted primer mix per set (4.0 nMfinal concentration per primer). Template materials (extracted RNAs, RNArunoff material, and RNA product from previous NASBA reactions) for eachprimer set were pooled and diluted and used as template for the boosterreaction. The template material was bound onto the Xtra Bind material inan Xtra Amp™ tube as above. This reaction was run at 40° C. for 15minutes and 30 minutes. The products from these reactions were dilutedten-fold and used as template for the ten separate, secondary simplexNASBA reactions.

The results of the sequential booster Xtra Plex NASBA reaction ofsolid-phase captured nucleic acid, shown in Table 3, indicate that eightof the ten NASBA systems worked. One of the failures was the same systemas in the above experiment. Again, the results are noteworthy in that atthe time of this writing, even multiplexing two distinct primer sets ina NASBA reaction has not been demonstrated.

TABLE 3 Sequential Booster Xtra Plex NASBA Reaction of Solid-PhaseCaptured Nucleic Acid Results Primer Pair Agarose Gel Results LateralFlow Results Group 1: E. coli SZ Negative Positive E. coli SLT 2Negative N/A L. monocytogenes HlyA Positive Positive C. trachomatis 16sPositive Positive E. coli LacZ 2 Positive Positive Group 2: E. coli SLT1 Negative Positive E. coli 16s Positive N/A N. gonorrhea 16s PositivePositive E. coli LacZ 1 Positive Negative E. coli UIDA Positive Negative10 Primer Pair NASBA Boosted Simplex Reactions E. coli SZ NegativeNegative E. coli SLT 2 Negative N/A L. monocytogenes HlyA PositivePositive C. trachomatis 16s Positive Positive E. coli LacZ 2 PositivePositive E. coli SLT 1 Negative Positive E. coli 16s Positive N/A N.gonorrhea 16s Positive Positive E. coli LacZ 1 Positive Negative E. coliUIDA Positive Negative

Example 3 Detection of E. Coli Using Sequences that Uniquely Identify E.Coli

The E. coli gene lacZ coding for beta-galactosidase (EC 3.2.1.23) wasfor identifying sequences that uniquely identify E. coli. Varioussequences of use in the detection of E. coli were located between basepairs 1700 to 1950 of the LacZ gene, identified as SEQ ID NO: 100 andshown below. Table 4 shows the lac Z gene sequences described by basepair number position relative to the E. coli genomic sequence in Genbank(accession VOO296, version VOO296.1 GI:41901).

SEQ ID NO: 100 TCTGGGACTGGGTGGATCAGTCGCTGATTAAATATGATGAAAACGGCAACCCGTGGTCGGCTTACGGCGGTGATTTTGGCGATACGCCGAACGATCGCCAGTTCTGTATGAACGGTCTGGTCTTTGCCGACCGCACGCCGCATCCAGCGCTGACGGAAGCAAAACACCAGCAGCAGTTTTTCCAGTTCCGTTTATCCGGGCAAACCATCGAAGTGACCAGCGAATACCTGTTCCGTCATAGCGATAACGA G

For specificity studies, nucleic acid was extracted onto Xtra Amp™ tubes(Xtrana, Inc, series III extraction kits) starting with high copy number(1,000,000 CFU) of various bacteria following a two hour induction with1 mM isopropyl β-D-thiogalactopyranoside (IPTG) by the inductionprotocol described in detail below. The extracted nucleic acid was thenNASBA amplified with various primer combinations. The specificity andspecific bacterial strains are summarized in Table 5. The sequencesconfirmed to be specific only to E. coli is primer set 5085/5086, thesequences of which are shown in Table 4. In addition, sequence 5085 withT7 promoter sequences added for NASBA (SEQ ID NO. 97) was used, whereSEQ ID NO: 97 is

5′AATTCTAATACGACTCACTATAGGGAGAGGACGGATAAACGGAACTGG A.Other primer pairs in this region were also specific to E. coli, withcertain individual strain exceptions as detailed in Table 5.

TABLE 4 starting ending Primer base base No. pair pair sense SEQUENCESEQ ID NO: 5085 1880 1897 (−) CGGATAAACGGAACTGGA 85 5086 1733 1750 (+)ATGATGAAAACGGCAACC 86 5100 1668 1687 (+) TCGTCAGTATCCCCGTTTAC 87 51011909 1928 (−) AGGTATTCGCTGGTCACTTC 88 5089 ^(a) 1856 1872 (−)CTGCTGGTGTTTTGCTT 89 5094 ^(a) 1761 1782 (+) TTACGGCGGTGATTTTGGCGAT 905064 ^(b) 1866 1880 (−) GAAAACTGCTGCTGG 91 5063 ^(b) 1681 1702 (+)CGTTTACAGGGCGGCTTCGTCT 92 5090 1866 1883 (−) GAAAAACTGCTGCTGGTGT 93 50971880 1900 (−) GCCCGGATAAACGGAACTGGA 94 5098 1721 1750 (+)CGCTGATTAAATATGATGAAAAC 95 GGCAACC 5099 1733 1755 (+)ATGATGAAAACGGCAACCCGTGG 96 ^(a) formerly LacZ 1 fp ^(b) formerly LaeZ 2fp

TABLE 5 E. coli E. E. C. K. S. S. Primer Pair Base Pairs spp. cloacaeaerogenes fruendii pneumoniae austin flexnerii 5085 + 5086 1733-1897 + —— — — NT — 5101 + 5100 1668-1928 + + — — — NT — 5090 + 50631681-1866 + + NT — — NT NT 5085 + 5063 1681-1897 + + NT — — NT NT 5097 +5098 1721-1900 + + NT — + NT NT 5063 + 5064 1681-1880 + + NT — — — NT5097 + 5086 1733-1900 + + NT — — NT NT 5097 + 5099 1721-1900 + + NT — —NT NT

Induction and purification of lacZ mRNA from low copy cells diluted intowater from cultures of E. coli bacteria was performed as follows. E.coli bacteria were routinely cultured on either LB (10 g/L tryptone, 5g/L yeast extract, 10 g/L sodium chloride and 15 g/L agar) or TSA agar(15 g/L pancreatic digest of casein, S g/L enzymatic digest of soybeanmeal and 5 g/L sodium chloride, 15 g/L agar) medium at 37° C. Liquidcultures were routinely prepared in TSB (15 g/L pancreatic digest ofcasein and 5 g/L enzymatic digest of soybean meal, 5 g/L sodiumchloride).

Small scale induction of lacZ mRNA was performed using 10% tryptone and1 mM IPTG (isopropyl β-D-thiogalactopyranoside). A single colony of E.coli bacteria was inoculated into 3 mL of TSB culture medium in a 15 mLculture tube. The culture was grown overnight at 37° C. with shaking toapproximately 1×10⁹ colony forming units (cfu) per mL. The culture wasdiluted in 0.5×SSC buffer (7.5 mM sodium citrate (pH 7), 75 mM NaCl) andan aliquot containing one to approximately 10⁶ cfu was added to a 15 mLtube containing 500 μL 10% tryptone, 1 mM IPTG. The culture wasincubated in a 37° C. water bath with shaking for about 2 to 6 hours toinduce the lacZ mRNA target.

An aliquot of the induced culture (50 to 100 μL) was transferred to anXtraAmp® extraction tube. The culture was lysed with an equal volume ofeither XtraAmp® Series 2 lysis buffer (Xtrana, Inc.), or XtraAmp® Series3 lysis buffer (Xtrana, Inc.). The lysis buffer was mixed briefly withthe culture and incubated 10 minutes at room temperature. The liquid wasremoved from the tube, and the bound nucleic acid was washed twice with100 μL volumes of either nuclease free water or XtraAmp Series 1 washbuffer.

NASBA amplification of a portion of the lacZ nucleic acid molecule usingprimers uniquely specific for the E. coli lacZ gene was performed asfollows. For this specific example primers denoted herein as Primer No.5085 (SEQ ID NO: 85) and Primer No. 5086 (SEQ ID NO: 86) were used.Primer No. 5085 contains 49 residues, the last 18 of which bind to aportion of the lacZ nucleic acid sequence in E. coli serovars (includingenterotoxigenic strains, e.g. E. coli O157:H7). The first 31 residues ofPrimer No. 5085 contain nucleic acid information required for T7 RNApolymerase binding and transcription initiation. The nucleic acidsequence Primer No. 5086 contains 18 residues that specifically bind theE. coli lacZ nucleic acid sequence. Kit reagents provided in theNuclisens Basic Kit (Biomerieux product No. 285053) were combined toproduce two solutions: an enzyme solution, and a master mix solutioncontaining 70 mM potassium chloride and 1.25 μM each of Primer No. 5085and Primer No. 5086. Aliquots (15 μL) of the master mix were added tonucleic acid bound in XtraAmp) tubes. The tubes were placed in a nucleicacid thermocycler or suitable heating instrument and incubated 2 minutesat 65° C. The tubes were cooled to 40° C. and 5 μL of enzyme solutionwas added to each tube. The tubes were heated for an additional 90minutes at 40° C.

The aqueous solution following amplification was assayed by lateral flowas described below and as detailed in U.S. patent application Ser. No.09/705,043, filed Nov. 2, 2000, which is specifically incorporatedherein by reference. Lateral flow chromatographic detection results in avisible blue line on a nitrocellulose strip in the presence of the 230base pair nucleic acid amplification product of E. coli lacZ mRNA usingPrimer Nos. 5085 and 5086. The detection primer set denoted herein asDetection Primer Mix No. 5097 contained Primer Nos. 5087 and 5088 at1.25 μM each. The specific sequence of the detection probes were:

Primer No. 5087: (SEQ ID NO: 98) 5′-FITC-GGTCGGCTTACGGCGGTG-phosphatePrimer No. 5088: (SEQ ID NO: 99) 5′-CTGTATGAACGGITCTGGTCTTTG-Biotin.

Primer No. 5087 includes 18 residues of the E. coli lacZ nucleic acidsequence. The 5′ and 3′ ends of Primer No. 5087 are modified with FITC(fluorescein isothiocyanate) and phosphate, respectively. Primer No.5088 includes 23 residues of the E. coli lacZ nucleic acid sequence. The3′ portion of Primer No. 5088 was modified with a biotin molecule. A 10μL portion of the NASBA reaction (described in Example 2) wastransferred to a 0.5 mL tube. One μL of Detection Primer Mix No. 5088and 40 μL of lateral flow buffer (50 mM TrisCl (pH 8), 8 mM MgCl₂, 0.25%Triton X-100, 0.8% PEG-8000) were added to 0.5 mL tube. The tube wasmixed and incubated 1 minute at 95° C. The tube was cooled for 1 minuteat room temperature, and the entire solution was applied to the samplepad of a lateral flow detection laminate containing embedded detectionconjugates. The sample was allowed to wick through the lateral flowstrip for approximately 5 minutes. Results were recorded visually. Ablue-colored line indicated the presence of the lacZ nucleic acidsequence as illustrated in FIG. 4.

Using the above protocol following a two hours either with or withoutIPTG revealed a dramatic increase in detection sensitivity as a resultof induction of lacZ mRNA transcription.

The foregoing description is considered as illustrative only of theprinciples of the invention. The words “comprise,” “comprising,”“include,” “including,” and “includes” when used in this specificationand in the following claims are intended to specify the presence of oneor more stated features, integers, components, or steps, but they do notpreclude the presence or addition of one or more other features,integers, components, steps, or groups thereof. Furthermore, since anumber of modifications and changes will readily occur to those skilledin the art, it is not desired to limit the invention to the exactconstruction and process described above. Accordingly, all suitablemodifications and equivalents may be resorted to falling within thescope of the invention as defined by the claims that follow.

1.-24. (canceled)
 25. A method for amplifying multiple nucleic acidtargets from a low abundant nucleic acid sample, the method comprising:a) performing a first amplification step, wherein the nucleic acidsample comprising the target nucleic acids is amplified such that thetarget nucleic acids are increased by 100-1000 fold, and wherein thefirst amplification step comprises a multiplex amplification reactionusing a first set of at least five primer pairs, and wherein the firstamplification step results in a pre-amplified nucleic acid samplecomprising a plurality of first amplification products; b) diluting thepre-amplified nucleic acid sample by at least 1:5; and, c) performing asecond amplification step on the pre-amplified nucleic acid sample fromstep (a) using at least two primer pairs used in step (a).
 26. Themethod of claim 25, wherein the nucleic acid sample is irreversiblybound to a solid phase material.
 27. The method of claim 25, wherein thenucleic acid sample is in aqueous solution.
 28. The method of claim 25,wherein the concentration of the primers used in the first amplificationstep is at a concentration that is at least 50× less than theconcentration of the primer concentration of the primers used in thesecond amplification step.
 29. The method of claim 25, wherein thenumber of amplification cycles of the first amplification step is lessthan ten cycles.
 30. The method of claim 25, wherein the nucleic acidsample is DNA.
 31. The method of claim 25, wherein the nucleic acidsample is RNA.
 32. The method of claim 25, wherein the firstamplification step is accomplished using a polymerase chain reaction.33. The method of claim 25, wherein the second amplification step isaccomplished using a polymerase chain reaction.
 34. The method of claim25, wherein a first set of 15-40 primer pairs are used in step (a). 35.The method of claim 25, wherein the method further comprises a step fordividing the pre-amplified nucleic acid sample into at least twodistinct amplification reactions prior to the second amplification step.36. The method of claim 35, wherein, for the second amplification step,the at least two distinct amplification reactions each comprise adifferent primer pair.
 37. The method of claim 25, wherein the at leasttwo primers of the first set of at least five primer pairs of step (a)have melting temperatures that differ by at least 5 degrees.
 38. Themethod of claim 25, wherein each of the at least five primer pairs instep (a) is at a concentration that is about between 2-6 nM.